The Ras family comprised of KRAS, NRAS and HRAS are mutated in one quarter of all human cancers, encoding an oncogenic, constitutively active protein that promotes tumorigenesis. While pharmacologically inhibiting oncogenic Ras has been challenging, targeting druggable proteins activated by Ras is proving to be a promising strategy. In this regard, we discovered in the last grant cycle that oncogenic Ras activates the endothelial member of the Nitric Oxide Synthase (eNOS) family to promote tumorigenesis. Excitingly, much of the time and cost to create NOS inhibitors can be curtailed by repurposing the drug L-NAME, originally developed to inhibit NOS enzymes in cardiogenic and septic shock patients. Indeed, L-NAME reduced tumor growth and provided a survival advantage in different mouse models of oncogenic KRas cancers, including highly aggressive pancreatic cancer models. These data led to a clinical trial to inhibit eNOS with a hydrolyzed form of L- NAME. To enhance this anti-tumor activity, we performed a L-NAME synthetic screen with kinase inhibitors. We identified 32 compounds, including a number of EGFR tyrosine kinase inhibitors, which reduced the viability of pancreatic cancer cells only in the presence of L NAME. Hence, we propose in aim 1 to evaluate the potential of combining L-NAME with the identified kinase inhibitors for the treatment of RAS mutation-positive cancers. To develop future therapeutic strategies to more specifically target this pathway, we performed a cell-based, high-throughput screen for small molecular weight inhibitors with a preference for eNOS over nNOS and iNOS, identifying seven lead compounds. We now propose in aim 1 to also evaluate the potential of these novel eNOS inhibitors for the treatment of RAS mutation-positive cancers. Finally, how activation of eNOS promotes cancer was unknown. In this regard, we found that wild type Ras proteins are S- nitrosylated on C118 and activated by eNOS. Moreover, replacing wild type HRas or NRas with C118S mutant versions reduced the tumor growth of oncogenic KRas cancer cells. Given these results, we propose in aim 2 to test the hypothesis that activation of eNOS in oncogenic Ras-driven cancers promotes tumorigenesis by S-nitrosylating and activating wild type Ras proteins. To this end, we generated a mouse in which one of the wild type Ras genes, Kras, was engineered to contain the aforementioned C118S mutation. We will now evaluate whether this mutation inhibits the ability of oncogenic Ras to promote tumorigenesis in vivo.